The molecular regulation of the invasion of osteosarcoma (OS) remains elusive. Here, we reported significant lower level of miR-143 and significant levels of phosphorylated EGFR and MMP9 in the resected OS from the patients, compared to the adjacent normal tissue. Moreover, strong correlation was detected among these three factors. We thus hypothesized existence of a causal link, which prompted us to use two human OS cell lines to study the interaction of miR-143, MMP9, and activation of EGFR signaling. We found that EGF-induced EGFR phosphorylation in both lines activated MMP9, and consequently cancer invasiveness. Both an inhibitor for EGFR phosphorylation and an inhibitor for ERK1/2 phosphorylation significantly inhibited the EGF-induced activation of MMP9. Moreover, miR-143 levels did not alter by EGF-induced EGFR phosphorylation, while overexpression of miR-143 antagonized EGF-induced MMP9 activation without affecting EGFR phosphorylation. Taken together, our data suggest that miR-143 inhibits EGFR signaling through its downstream ERK/MAPK signaling cascades to control MMP9 expression in OS. Thus, miR-143, EGFR, and MMP9 are therapeutic targets for inhibiting OS invasion.